ChIP protocol (View forum version)



lsek

Posted 01 June 2009 - 09:19 PM

Hi Everyone,

I need help. I working on ChIP and was wondering if anyone is doing ChIP with solutions prepared by themselves.
Could you help confirm if the components and its composition are correct:

ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-Cl, pH 8.1)
ChIP dilution buffer (0.01%, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl)
Low salt immune complex [for wash step 1] (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 150 mM NaCl)
High salt immune complex [for wash step 2] (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 500 mM NaCl)
LiCl immune complex [for wash step 3] (0.25M LiCl, 1% IGEPAL Ca630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-Cl, pH 8.1)
TE buffer (10 mM Tris-Cl, pH 8.1, 1 mM EDTA)

Elution buffer (1% SDS, 0.1M NaHCO3) prepared fresh by adding from stocks of 10% SDS and 1M NaHCO3.

Sorry for the trouble. I'm trying to troubleshoot my ChIP experiment which is not working. Ohya, do I need to add protease inhibitors for the washing steps? Thank you so much for the help.

madrius1

Posted 02 June 2009 - 07:04 AM

I use homemade buffers, and my experiments work pretty well. Here, i'll compare to your buffers.

ChIP lysis : 1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1. Add 1mM PMSF and 1X protease inhibitor cocktail before use.
ChIP dilution buffer : 0.01% SDS, 1.1% Triton, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl. Store at 4C and add 1mM PMSF and 1X protease inhibitor cocktail to the required volume for the ChIP before use.
Wash 1 (TSE-150) : 0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris pH 8.1, 150 mM NaCl
Wash 2 (TSE-500) : 0.1% SDS, 1 % Triton, 2 mM EDTA, 20 mM Tris pH 8.1, 500 mM NaCl.
LiCl : 0.25 M LiCl, 1% NP40, 1% DOC, 1 mM EDTA, 10 mM Tris pH 8.1.

TE and Elution are the same. So your buffers are the same as mine!

And no, you don't need protease inhibitors in the washing steps.

lsek

Posted 04 June 2009 - 08:32 AM

Thank you so much! :o

Sorry for the late reply.

Cheers.